DNA-affinic hypoxia selective cytotoxins

ABSTRACT

Hypoxia selective cytotoxins having the structural formula ##STR1## wherein D, E, F and G, independently, are carbon or nitrogen, with the proviso that three or more of D, E, F and G are carbon; R 1  and R 2 , independently, are selected from the group consisting of methyl, halo, hydro, trifluoromethyl, methoxy, cyano, and methylsulfo; R 3  and R 4 , independently, are selected from the group consisting of methyl, ethyl, phenyl, naphthyl, tertiary butyl, halo, halomethylene, hydro, trifluoromethyl, cyano and methylsulfo, or R 3  and R 4  taken together are a substituted or unsubstituted five or six-membered nonaromatic ring system; n is an integer 1 through 5; X is carbon or nitrogen; and Z is a physiologically acceptable anion, are disclosed. The compounds are useful as radiosensitizers or chemosensitizers, especially in the treatment of cancer patients.

FIELD OF THE INVENTION

The present invention generally relates to cancer therapy and tocompounds and methods of sensitizing tumor cells to radiation therapyand chemotherapy. More particularly, the present invention relates to anovel class of DNA-affinic hypoxia selective cytotoxins whichradiosensitize and chemosensitize hypoxic tumor cells.

BACKGROUND OF THE INVENTION

Malignant tumors often demonstrate a resistance to radiation therapy andchemotherapy. The resistance of physiologically hypoxic regions withinsolid tumors during cancer treatment is an important reason for thefailure of radiotherapy and chemotherapy to eradicate such tumors.

Investigators have made progress in understanding the basic cellular andmolecular mechanisms of therapeutic resistance. Many of theseinvestigative efforts have focused on intrinsic cellularcharacteristics. However, there is an aspect of therapeutic resistancewhich is related to the physiologic and biochemical state of the cell,and not to intrinsic cellular properties. In other words, cells whichare otherwise sensitive to cytotoxin treatment under normal physiologicconditions, are resistant because of their particular physiologic statewithin the tumor.

For example, it has long been known that hypoxic, i.e., oxygendeficient, cells are relatively resistant to killing by radiation. Toachieve the same proportion of cell kill, about three times theradiation dose is required for hypoxic cells compared to the radiationdose required for well-oxygenated cells. Thus oxygen has the ability tosensitize cells to ionizing radiation at clinical radiation doses.Overcoming this resistance of hypoxic cells has been investigated as ameans of improving the efficacy of ionizing radiation.

Multiple mechanisms have been proposed to explain hypoxic resistance toradiation therapy and chemotherapy. The proposed mechanisms involvekinetic, metabolic, and physical factors. For example, hypoxic cellsfrequently are noncycling and therefore are refractory toproliferation-dependent cytotoxic drugs. In addition, the cell can be ina metabolically-compromised state and unable to concentrate and activatepotentially-effective agents. The distance between a cell and bloodvessels also can be greater than the diffusion distance of manychemotherapeutic agents.

Efforts to overcome hypoxia in clinical cancer treatments have involvedthe development of hypoxic cell radiosensitizers and chemosensitizerswhich substitute for, or mimic, oxygen.

Cell kill by ionizing radiation is caused by damage to the DNA. Thetarget radical on the DNA, designated "DNA-", is produced by eitherdirect ionization or reaction with hydroxyl radicals produced radiolysisof neighboring water molecules. Reaction with oxygen produces a peroxylradical, DNA-O₂., which forms products leading to irreversible DNAdamage. Radiosensitizers are designed to mimic oxygen by reacting withDNA radicals to form covalent adducts at the radical sites.

As discussed hereafter, the ability of oxygen or a sensitizer to enhancecell kill is reflected in the enhancement ratio, i.e., OER for oxygenand SER (sensitizer enhancement ratio) for the sensitizer. The OER isdependent on the concentration of oxygen present at the target at thetime of irradiation. Similarly, the oxygen-mimicking effect of a hypoxiccell sensitizer (SER) depends primarily on the concentration ofsensitizer at the target at the time of irradiation. However, theoxygen-mimicking sensitizers preferentially affect hypoxic cells, whichtypically comprise only about 20% of the tumor. Therefore, in estimatingthe degree of enhancement of cell kill, the SER applies only to hypoxiccells, not to the entire tumor.

It is well established that bioreductive compounds, such asnitroimidazole-based compounds, potentiate the cytotoxic effects ofradiation and several chemotherapeutic agents towards hypoxic tumorcells, both in vitro and in vivo. Bioreductive compounds are readilyactivated by metabolic reduction in a hypoxic environment and enhancethe susceptibility of hypoxic tumor cells to radiation and conventionalanticancer drugs.

Bioreductive agents typically are compounds of high electron affinity.Bioreductive agents have the ability to kill hypoxic cells directlybecause of their preferential reductive metabolism under hypoxicconditions, where the limited oxygen concentration cannot significantlyantagonize the reduction process. In addition, solid tumors developphysiological hypoxia to a greater degree than normal tissues, andevidence exists that tumor cells have relatively high levels ofreductive enzymes.

Bioreductive agents in hypoxic cells therefore mimic the oxygen effectin oxygenated cells during irradiation, and cause fixation ofradiation-induced damage to DNA or other vital macromolecules. Thus,bioreductive agents act as radiosensitizers of hypoxic cells.Bioreductive agents can also act as chemosensitizers for conventionalanticancer drugs by enhancing the susceptibility of hypoxic tumor cellsto chemotherapy.

The combination of a sensitizer with either radiation or a conventionalanticancer drug helps overcome the problem of hypoxic cell resistance tocancer therapy. Chemosensitization in vitro usually is demonstrated bypretreating cells with a sensitizer under hypoxic conditions beforeexposure to the chemotherapeutic drug, often an alkylating agent, underaerobic conditions. This "preincubation effect" is attributedpredominantly to a reduction of the sensitizer which occurs underhypoxic conditions.

Investigators have searched for improved hypoxic cell sensitizers thatare non-toxic to aerobic and that concentrate more effectively intumors. One hypoxic cell radiosensitizer is misonidazole (MISO), anelectron-affinic 2-nitroimidazole which has shown some benefit incertain situations. However, MISO exhibits significant neurotoxicityand, consequently, the total dose of MISO that can be administered to apatient is limited. Another hypoxic cell sensitizer is etanidazole(SR-2508), a neutral compound which is more hydrophilic than MISO, isless neurotoxic, and can be administered to humans at about a threefoldhigher dose than MISO.

A third hypoxic cell sensitizer is pimondazole (Ro 03-8799), whichcontains a basic piperidine moiety and has a total dose limitationsimilar to MISO. A fourth compound, RSU-1069, is a bifunctional moleculecontaining a 2-nitroimidazole group and an alkylating aziridine. Inexperimental systems, RSU-1069 has a substantially greater activity thanMISO, and is toxic to hypoxic cells in vitro at about a 100-fold lowerconcentration compared to the toxic concentration for aerobic cells.

Bioreductive radiosensitizers also have an ability to significantlyenhance the activity of several chemotherapeutic agents, such as, e.g.,cyclophosphamide, nitrosoureas, L-phenylalanine mustard (i.e., L-PAM or4-[bis(2-chloroethyl)amino]-L-phenylalanine),cis-diamminedichloroplatinum(II) (i.e., cis-DDP) and doxorubicin, invitro and in vivo. This enhancement of chemotherapeutic activity isknown as chemosensitization or chemopotentiation.

Although significant progress has been made in developing bioreductivedrugs as radio- and chemosensitizers, further development is necessarybecause none of the bioreductive drugs tested to date has shownimpressive clinical results. Currently, therefore, there is a stronginterest in targeting bioreductive agents to DNA in order to improve theradio- and chemosensitizing properties of such agents. Efforts toincrease cytotoxic efficacy have centered on increasing theconcentration of sensitizer within DNA as opposed to increasing theaverage intracellular concentration. Some investigators targeted DNA bycombining an alkylating agent with bioreductive functional groups withinthe same drug (e.g., RSU-1069). Other investigators used transitionmetal coordination complexes such as platinum and ruthenium to targetnitroaromatic radiosensitizers to DNA. However, these metal coordinationcomplexes often are less effective as radiosensitizers than the freeradiosensitizer molecule, even though the one electron reductionpotential (a property related to sensitization efficiency) can beincreased in some platinum complexes compared to the free sensitizermolecule.

Another approach to improve targeting of bioreductive agents to DNAinvolves using an intercalating moiety such as a phenanthridine or anacridine, which inserts itself between two adjacent sets of base pairsof the DNA. Non-covalent binding to DNA, such as through intercalation,permits migration of the radiosensitizer to DNA sites where radiationinduced radicals are created.

For example, NLP-1, 5-[3-(2-nitro-1-imidazolyl)-propyl]phenanthridiniumbromide, a 2-nitroimidazole-linked phenanthridine, has been synthesized.The synthesis, hypoxic cell cytotoxicity and radiosensitization of NLP-1has been reported by R. Panicucci et al., Int. J. Radiat. Oncol. Biol.Phys., 16, pages 1039-1043 (1989), incorporated herein by reference.##STR2##

An acridine-based hypoxia selective cytotoxin is preferred over thephenanthridine-based compound because acridine is a better intercalatorthan phenanthridine. Nitracrine, 1-nitro-acridine, is a potent hypoxiaselective cytotoxin and a radiation sensitizer in mammalian cellcultures; however, rapid metabolism limits the radiosensitizationefficacy of nitracrine in vivo. See, P. B. Roberts et al., RadiationResearch, 123, pages 153-164 (1990), incorporated herein by reference.

An acridine-based hypoxia selective cytotoxin that is relatively stablein vivo therefore is preferred. In addition, it is preferred that thehypoxia selective cytotoxin does not bind tightly to DNA. For example,1-nitracrine, which exhibits faster dissociation kinetics from DNA thanthe other nitroacridine isomers, is twenty times more potent as asensitizer than other nitracrine isomers tested.

Papadopoulou-Rosenzweig et al. U.S. Pat. No. 5,294,715, incorporatedherein by reference, discloses hypoxia selective cytotoxins having thestructure ##STR3## wherein n is from 1 to 5, and NO₂ is in at least oneof the 2, 4 or 5-positions of the imidazole ring.

The compounds disclosed in Papadopoulou-Rosenzweig et al. U.S. Pat. No.5,294,715 include an aromatic acridine moiety and are useful asradiosensitizers and chemosensitizers. These acridine-based compoundsare electron affinic and exhibit strong DNA intercalating properties.However, the acridine-based compounds demonstrated a less than expectedradiosensitization in vivo. This unexpectedly low radiosensitization hasbeen attributed to restricted mobility of the acridine-based compoundsalong the DNA backbone, and to low extravascular diffusion in tumors.Mobility along the DNA backbone is considered a significant factor withrespect to trapping radiation-induced radicals and providing goodradiosensitization and chemosensitization efficacy.

Investigators therefore have continued efforts to develop a hypoxiaselective cytotoxin and sensitizer having a lower affinity to bind toDNA and having enhanced efficacy. Accordingly, the present invention isdirected to bioreductive cytotoxins which enhance the cytotoxicactivities of ionizing radiation and chemotherapeutic agents to hypoxiccells, which are inherently cytotoxic to hypoxic cells, and which areessentially nontoxic to aerobic cells.

SUMMARY OF THE INVENTION

The present invention is directed to a novel class of compounds that areDNA affinic and that exhibit substantial mobility along the DNAbackbone. The compounds are useful as sensitizers in radiotherapy andchemotherapy. In particular, the present invention is directed to anovel class of compounds which act as hypoxia selective cytotoxins andwhich do not exhibit significant aerobic toxicity at effective radio-and chemosensitization doses.

Therefore, one aspect of the present invention is to providebioreductive compounds having radiosensitization and chemosensitizationproperties. The compounds are capable of binding to DNA byintercalation, and exhibit a significant mobility along the DNAbackbone. The compounds also are more selectively toxic to hypoxic cellsthan to aerobic cells. The novel hypoxia selective compounds are usefulin methods of treating cancer by potentiating the toxic effect ofradiation and chemotherapy. The present hypoxia selective compounds alsoexhibit improved radiosensitization and chemosensitization over priorsensitizers.

In particular, the present invention is directed to hypoxia selectivecytotoxins having general structural formula (I): ##STR4## wherein D, E,F and G, independently, are carbon or nitrogen, with the proviso thatthree or more of D, E, F and G are carbon; R₁ and R₂, independently, areselected from the group consisting of methyl, halo, hydro (i.e., H),trifluoromethyl, methoxy, cyano, and methylsulfo; R₃ and R₄,independently, are selected from the group consisting of methyl, ethyl,tertiary butyl, phenyl, naphthyl, halo, halomethylene, hydro,trifluoromethyl, cyano, and methylsulfo, or R₃ and R₄ taken together area substituted or unsubstituted five or six-membered nonaromatic ringsystem; n is an integer 1 through 5; X is carbon or nitrogen; and Z is aphysiologically acceptable anion.

In another aspect of the present invention, the preferred hypoxiaselective cytotoxins have R₃ and R₄ groups that are taken together toform a substituted or unsubstituted five or six-membered nonaromaticring system. Exemplary hypoxia selective cytotoxins of the presentinvention therefore include, but are not limited to,9-[3-(2-nitro-1-imidazolyl)propylamino]-1,2,3,4-tetrahydroacridinehydrochloride (THNLA-1),10-[3-(2-nitroimidazolyl)propylamino]-3,4-dihydro-1-H-thiopyrano[4,3-b]quinolinehydrochloride (S-THNLA-1), 10-[3-(2-nitroimidazolyl)propylamino]-2-methyl-1,2,3,4-tetrahydro-benzo[b]-1,6-naphthyridinehydrochloride (MeN-THNLA-1) and9-[3-(2-nitro-1-imidazolyl)propylamino]cyclopenten[b]quinolinehydrochloride (NLCPQ-1), having the structural formulae ##STR5##

In another aspect of the present invention, in addition to the fusedaromatic ring system including two rings (e.g., the quinoline ringsystem), the present hypoxia selective cytotoxins also include anonplanar, nonaromatic fused ring, or other suitable substituent orsubstituents, bonded to the b-ring of the aromatic fused ring system toincrease the mobility of the hypoxia selective cytotoxin along the DNAbackbone by decreasing the binding efficacy to DNA.

Another aspect of the present invention is to provide a method ofradiosensitization comprising administering an effective amount of thehypoxia selective cytotoxin of structural formula (I), thenadministering ionizing radiation.

Another aspect of the present invention is to provide a method ofchemosensitization comprising administering an effective amount of ahypoxia selective cytotoxin of general formula (I), then administering achemotherapeutic agent.

Another aspect of the present invention is to provide a method ofradiosensitization comprising administering an effective dose ofTHNLA-1, S-THNLA-1, MeN-THNLA-1 or NLCPQ-1, then administering ionizingradiation.

Another aspect of the present invention is to provide a method ofchemosensitization comprising administering an effective dose ofTHNLA-1, S-THNLA-1, MeN-THNLA-1 or NLCPQ-1, then administering achemotherapeutic agent.

Yet another aspect of the present invention is to provide a method oftargeting a hypoxia selective cytotoxin to the DNA of hypoxic tumorcells comprising treatment with compounds having general structuralformula (I).

The present compounds are particularly well suited as hypoxia selectivecytotoxins due to the feature of a nitroimidazole moiety as thehypoxiaselective moiety. In accordance with an important aspect of thepresent invention, the nitroimidazole moiety is linked to aquinoline-based, fused aromatic ring system through an alkylamino chain.The compounds effectively target the sensitizing moiety to DNA throughintercalation. In a preferred form, the present compounds are positivelycharged to increase sensitizer concentration near the negatively-chargedphosphate moieties present in the DNA backbone. The present compoundsfurther feature a nonplanar, nonaromatic ring, or other suitablesubstituents, bonded to the b-ring of the quinoline ring system. Thenonplanar nonaromatic ring, or other substituents, increase the mobilityof the hypoxia selective cytotoxin along the DNA backbone due to weakerbinding to DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects and advantages of the present invention willbecome apparent from the following detailed description of the preferredembodiments illustrated in the accompanying figures wherein:

FIG. 1A is a plot of survival fraction of V79 Chinese hamster lung cellsvs. time for a constant 0.1 mM (millimolar) concentration of THNLA-1under aerobic or hypoxic conditions;

FIG. 1B is a plot of survival fraction of V79 cells vs. THNLA-1concentration after a one hour time period at 37° C. under aerobic orhypoxic conditions;

FIGS. 2A-C are plots of survival fraction of V79 cells vs. S-THNLA-1,MeN-THNLA-1 or NLCPQ-1 concentrations, respectively, after a one hourtime period at 37° C. under aerobic or hypoxic conditions;

FIG. 3A is a plot of survival fraction of hypoxic or aerobic V79 cellsvs. radiation doses for various THNLA-1 concentrations;

FIG. 3B is a plot of SER (sensitization enhancement ratio) vs. THNLA-1concentration;

FIG. 4 is a plot of SER vs. THNLA-1, S-THNLA-1 and MeN-THNLA-1concentration;

FIGS. 5A and 5B are plots of survival fraction vs. concentration ofTHNLA-1 or NLA-1 at a radiation dose of 7.5 Gy or 20 Gy under hypoxic oraerobic conditions;

FIGS. 6A and 6B are plots of survival fraction vs. concentration ofNLA-1, THNLA-1 and S-THNLA-1 at a radiation dose of 7.5 Gy or 20 Gyunder aerobic or hypoxic conditions;

FIG. 7A is a plot of intracellular (C_(i)) and extracellular (C_(e))concentrations of THNLA-1 vs. THNLA-1 input concentration;

FIG. 7B is a plot of C_(i) /C_(e) (uptake factor) vs. THNLA-1 inputconcentrations under aerobic conditions for 30 minutes at 37° C.;

FIG. 7C is a plot of THNLA-1 uptake (C_(i) and C_(e)) by V79 cells underaerobic or hypoxic conditions vs. time for a constant 20 μM (micromolar)concentration of THNLA-1 at 37° C.;

FIG. 8 is a plot of C_(i) and C_(e) vs. input concentrations of THNLA-1,S-THNLA-1, MeN-THNLA-1 and NLCPQ-1;

FIGS. 9A and 9B are plots of C_(e) and C_(i) (μM) of THNLA-1 and NLA-1,respectively, at 60 μM input concentration in aerobic V79 cells vs.ammonium chloride concentration (mM);

FIG. 9C is a plot of C_(i) vs. ammonium chloride concentration comparingthe effect of ammonium chloride on C_(i) of THNLA-1 and NLA-1 in aerobicV79 cells;

FIG. 10A is a plot of C_(e) and C_(i) (μM) of S-THNLA-1 (measured at330nm) vs. NH₄ Cl concentration;

FIG. 10B is a plot of C_(i) (measured at 330nm) vs. NH₄ Cl concentrationcomparing the effect of NH₄ Cl on intracellular accumulation of THNLA-1,S-THNLA-1 and NLA-1 in aerobic V79 cells;

FIG. 11A is a plot of uptake reduction rates for NLA-1 and THNLA-1 (inμM per mM of NH₄ Cl ) vs. ammonium chloride concentration;

FIG. 11B is a plot of hypoxia selective cytotoxin concentrationnecessary for an SER of 1.6 vs. C_(i) /C_(e) for various hypoxiaselective cytotoxins;

FIGS. 12A and 12B are plots of survival fraction of V79 cells vs.hypoxia treatment time in the presence of THNLA-1, L-PAM or cis-DDP, acombination of THNLA-1 and L-PAM or cis-DDP, and the survival fractionexpected from a combination of THNLA-1 and L-PAM or cis-DDP;

FIGS. 13A and 13B are plots of survival fraction of V79 cells vs.THNLA-1 concentration (μM), wherein THNLA-1 is present alone or in thepresence of L-PAM or cis-DDP, also plotted is the survival fractionexpected from a combination of THNLA-1 and either L-PAM or cis-DDP;

FIGS. 14A and 14B are plots of survival fraction of V79 cells vs. L-PAM(μg/ml) or cis-DDP (μM) concentration, wherein L-PAM or cis-DDP ispresent alone or in combination with THNLA-1;

FIGS. 15A and 15 B are isobolograms plotting THNLA-1 concentration vs.L-PAM (μM) or cis-DDP (μM) concentrations showing the zones ofsynergistic, additive and antagonistic effects, and that experimentaldata of the combination treatment with THNLA-1 and L-PAM or cis-DDPfalls within the synergistic zone;

FIGS. 16A and 16B are plots of survival fraction vs. time of THNLA-1administration (0.103 mmol/g) before irradiation (min) for replicatetests performed on tumor-bearing mice to determine the radiosensitizingeffects of THNLA-1 in vivo; and

FIGS. 17A-C are plots of survival fraction vs. time of etanidazoleadministration (2 mmol/g) before irradiation (min) for replicate testsperformed on tumor-bearing mice to determine the radiosensitizingeffects of etanidazole in vivo.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

By directing an electron-affinic compound to its expected site ofaction, i.e., DNA, the potency of the compound as a radiosensitizer andchemosensitizer, as well as its hypoxic cell cytotoxicity, is greatlyincreased. Since mobility of the compound along the DNA backbone also issignificant with respect to trapping radiation-induced radicals, andtherefore improving radiosensitization, the development of DNA-affiniccompounds that bind to DNA through non-covalent mechanisms (e.g.,intercalation) is desirable.

However, compounds that bind non-covalently, but strongly, to DNA haveslow dissociation kinetics. These compounds are potent cytotoxinsbecause of mechanisms independent of bioreductive activation, e.g., byhindering the movement of polymerases or interfering with the action oftopoisomerases I/II along the DNA backbone. Slow DNA dissociationkinetics can result in low hypoxic selectivity. Slow DNA dissociationkinetics can also be responsible for restricted extravascular diffusionof DNA-affinic compounds to hypoxic regions of tumors, thereforelimiting the effectiveness of strong DNA-affinic compounds in vivo.

Thus, new DNA-affinic bioreductive compounds having greater mobilityalong the DNA backbone have been prepared. These new compounds exhibitimproved radiosensitizing and chemosensitizing effectiveness, and animproved selective cytotoxicity towards hypoxic cells in vitro and invivo. In general, the compounds of the present invention also exhibitimproved therapeutic indices over prior sensitizers.

As discussed hereafter, it has been hypothesized, but not relied uponherein, that the improved radiosensitizing and chemosensitizing effectsdemonstrated by the present compounds are a result of disrupting theplanarity of aromatic ring systems that are present in priorsensitizers, such as the substituted acridines disclosed inPapadopoulou-Rosenzweig et al. U.S. Pat. No. 5,294,715. The presenthypoxia selective cytotoxins have a lower DNA-binding affinity than theacridine-based compounds because the present compounds are based: (1)either on a quinoline or quinoline-related aromatic system havingsubstituents or (2) a nonaromatic ring system bonded to the b-ring ofthe quinoline or quinoline-related aromatic fused-ring system. Inaddition, the present compounds are weak bases having a greaterlipophilicity than the acridine-based compounds, and therefore alsoexhibit enhanced cellular uptake for greater efficacy.

As also discussed hereafter, the present DNA-affinic compounds exhibitan improved hypoxia selective cytotoxicity and an improvedradiosensitization and chemosensitization over the acridine-basedcompounds disclosed in Papadopoulou-Rosenzweig et al. U.S. Pat. No.5,294,715 presumably because the present compounds intercalate lessstrongly with DNA. It has been found that the sensitizing efficacy andhypoxia selective cytotoxicity of compounds can be improved by reducing(i.e., hydrogenating) or eliminating the third aromatic ring of theacridine-based sensitizers. This improvement is attributed tointercalating DNA less strongly, therefore increasing the dissociationkinetics with DNA, and without influencing the appropriate one-electronreduction potential values. The present class of bioreductive agentstherefore provides better adjuvant therapy in both radiotherapy andchemotherapy for solid tumor treatment.

In particular, the present invention is directed to a novel class ofhypoxia selective cytotoxins and their use in radiation therapy andchemotherapy as sensitizers. In particular, hypoxia selective cytotoxinsof the present invention have a general structural formula (I): ##STR6##wherein D, E, F and G, independently, are carbon or nitrogen, with theproviso that three or more of D, E, F and G are carbon; R₁ and R₂,independently, are selected from the group consisting of methyl, halo,hydro, trifluoromethyl, methoxy, cyano and methylsulfo; R₃ and R₄,independently, are selected from the group consisting of methyl, ethyl,tertiary butyl, phenyl, naphthyl, halo, halomethylene (e.g., FCH₂₋),hydro, trifluoromethyl, cyano and methylsulfo, or R₃ and R₄ takentogether are a substituted or unsubstituted five or six-memberednonaromatic ring system; n is an integer 1 through 5; X is carbon ornitrogen; and Z is a physiologically acceptable anion.

Compounds having general structural formula (I) therefore have a fusedring system including two aromatic rings, wherein a nitrogen atom ispresent in at least one of the rings. The compounds of generalstructural formula (I) therefore are based on the structure ofquinoline, which has structural formula (II): ##STR7## The benzo-ring istermed the a-ring of quinoline. The pyrido-ring is termed the b-ring ofquinoline.

In addition to quinoline, other quinoline-related fused aromatic systemscan be utilized in the present invention. Accordingly, D, E, F and G ofthe compound of general structural formula (I) can be, independently,carbon or nitrogen, provided that three or more of D, E, F and G arecarbon. It should be understood that if D, E, F, or G is nitrogen, thenthe nitrogen is unsubstituted. In addition, if D, E, F, or G is carbon,and if R₁ or R₂ is not present on that carbon atom, then that carbon isbonded to a hydrogen atom.

Exemplary quinoline-related ring systems that can be used in addition toquinoline include, but are not limited to, fused ring systems having astructural formula (III) through (VI): ##STR8##

In accordance with an important feature of the present invention, R₁ andR₂ can be any organic substituent group that does not adversely affectthe hypoxia selective cytotoxicity, radiosensitizing capabilities orchemosensitizing capabilities of the compound of general structuralformula (I). Accordingly, R₁ and R₂, independently, can be, but are notlimited to, hydro, i.e., hydrogen; methyl; halo, i.e., chloro, bromo,iodo or fluoro; trifluoromethyl; methoxy; cyano; or methylsulfo.Preferred R₁ and R₂ groups are hydro, chloro, fluoro, methyl, methoxy,and trifluoromethyl.

Substituent groups R₃ and R₄ bonded to the nitrogen-containing b-ring ofthe quinoline or quinoline-related aromatic fused ring system can be forexample, but are not limited to, hydro; methyl; ethyl; tertiary butyl;phenyl; naphthyl; halomethylene; halo, i.e., chloro, bromo or iodo;cyano; and methylsulfo.

In addition, R₃ and R₄ can be taken together to form a substituted orunsubstituted five or six-membered nonaromatic ring system. Thenonaromatic ring system can include heteroatoms, such as sulfur, oxygenor nitrogen. In nonaromatic ring systems including a nitrogen atom, thenitrogen atom has a methyl or ethyl group as a substituent.

Exemplary ring systems derived from combining substituents R₃ and R₄include, but are not limited to, the nonaromatic ring systemsillustrated in structural formulae (VII)-(XIV): ##STR9## wherein R₅ isan alkyl group having one or two carbon atoms (i.e., methyl or ethyl).

Preferred R₃ and R₄ groups are hydro, halo, methyl, ethyl, phenyl,naphthyl, and trifluoromethyl. To achieve the full advantage of thepresent invention, R₃ and R₄ are taken together to form a nonaromaticfive or six-membered ring, either carbocyclic or incorporating an oxygenatom, a sulfur atom or a nitrogen atom.

In accordance with an important feature of the present invention, thefollowing are preferred combinations of the R₁ and R₂ substituents:

    ______________________________________                                                R.sup.1                                                                             R.sup.2                                                         ______________________________________                                                H     H                                                                       CF.sub.3                                                                            H                                                                       CH.sub.3                                                                            H                                                                       CH.sub.3                                                                            CH.sub.3                                                                F     H                                                                       F     F                                                                       CH.sub.3 O                                                                          H                                                                       CH.sub.3 O                                                                          CH.sub.3 O                                                              Cl    H                                                                       Cl    Cl                                                              ______________________________________                                    

Similarly, the following are preferred combinations of the R₃ and R₄substituents:

    ______________________________________                                                R.sup.3                                                                            R.sup.4                                                          ______________________________________                                                H    H                                                                        H    CH.sub.3                                                                 CH.sub.3                                                                           CH.sub.3                                                                 H    CH.sub.3 CH.sub.2                                                        H    CF.sub.3                                                                 H    CMe.sub.3                                                                H    Naph                                                                     H    Cl                                                                       H    Ph                                                               ______________________________________                                    

wherein CMe₃ is tert-butyl, Naph is naphthyl and Ph is phenyl.

In accordance with another important feature of the present invention,the following are preferred nonaromatic ring systems when R₃ and R₄ aretaken together to form a nonaromatic five or six-membered ring:

--(CH₂)₃ --

--CHMe(CH₂)₂ --

--CH₂ CHMeCH₂ --

--(CH₂)₂ CHMe--

--(CH₂)₄ --

--CHMe(CH₂)₃ --

--CH₂ CHMe(CH₂)₂ --

--(CH₂)₂ CHMeCH₂ --

--(CH₂)₃ CHMe--

--SCH₂ CH₂ --

--CH₂ SCH₂ --

--CH₂ SCH₂ CH₂ --

--CH₂ OCH₂ --

--CH₂ OCH₂ CH₂ --

--CH₂ NR₅ CH₂ CH₂ --

--(CH₂)₃ NR₅ --

--(CH₂)₂ NR₅ --,

wherein Me is methyl and R₅ is methyl or ethyl.

In the compound of general structural formula (I), the fused aromaticring system is linked to a five-membered, nitrogen-containing aromaticheterocyclic ring by a methylene chain including one to five (i.e., n isan integer one through five) methylene (i.e., CH₂) groups. Preferably,the number of methylene groups is two to four, i.e., n is an integer twothrough four. The five-membered nitrogen-containing aromaticheterocyclic ring is based on imidazole and has one nitro group (NO₂) asa substituent. Imidazole has the structure (XV): ##STR10## and isdepicted in the compound of general structural formula (I) when X iscarbon.

In addition to imidazole, however, another nitrogen-containing,five-membered aromatic ring also is useful in the present invention, asdepicted in the compound of structural formula (I) when X is nitrogen.This five-membered aromatic ring is depicted in structural formula (XVI). ##STR11##

The hypoxia selective cytotoxins of general structural formula (I) aresalts provided by adding an acid to the neutral compound. The salt formof the present compounds is depicted in structural formula (I).Conventionally, the salt form is used to increase sensitizerconcentration along the DNA-backbone because of electrostatic attractionbetween the positively-charged compound of structural formula (I) andthe negatively-charged phosphate moieties of DNA. The salt form is usedto facilitate administration of the compound of structural formula (I)because the salt is more soluble in water or saline than the neutralform of the compound.

However, the neutral form of the compound depicted in general structuralformula (I) also is useful as a hypoxia selective cytotoxin and as aradio-or chemosensitizer. The neutral form of the present compounds alsocan be used to intercalate with DNA and therefore act as sensitizers.The neutral form of the present compounds is protonated in vivo afteradministration to provide the salt form of the compound. The neutralform of the compound of general structural formula (I) is protonated atphysiological pH (e.g., about 7) because the compounds are weak baseshaving a pK_(a) of about 9.5 to about 10. The neutral form of thepresent class of hypoxia selective cytotoxins has general structuralformula (XVII): ##STR12## wherein D, E, F, G, X, Y, n and R₁ -R₄ havethe definitions previously set forth.

When preparing a compound of general structural formula (I), the neutralcompound of structural formula (XVII) first is prepared. The neutralcompound of structural formula (XVII) then is neutralized with an acidhaving a physiologically-acceptable anion. Accordingly, the acid (i.e.,HZ) can be, but is not limited to, hydrochloric acid, phosphoric acid,nitric acid, perchloric acid, tetrafluoroboric acid, sulfuric acid, or amixture thereof. Accordingly, the component Z in structural formula (I)can be, but is not limited to, chloride, bisulfate, dihydrogenphosphate, nitrate, perchlorate, tetrafluoroborate, or a mixturethereof.

Synthesis of exemplary novel compounds of the present invention isillustrated diagrammatically in the following schematic syntheticsequence which sets forth the method of synthesizing the compoundsTHNLA-1, S-THNLA-1, MeN-THNLA-1 and NLCPQ-1.

The following is a list of abbreviations used hereafter:

THNLA-1:9-[3-(2-nitro-1-imidazolyl)propylamino]-1,2,3,4-tetrahydroacridinehydrochloride;

S-THNLA-1:10-[3-(2-nitroimidazolyl)propylamino]-3,4-dihydro-1-H-thiopyrano[4,3-b]quinolinehydrochloride;

MeN-THNLA-1:10-[3-(2-nitroimidazolyl)propylamino]-2-methyl-1,2,3,4-tetrahydro-benzo[b]-1,6-naphthyridinehydrochloride;

NLCPQ-1: 9-[3-(2-nitroimidazolyl)propylamino]-cyclopenten[b]quinolinehydrochloride;

NLA-1: 9-[3-(2-nitro-1-imidazolyl)propylamino]acridine hydrochloride;

NaI: sodium iodide;

PhOH: phenol;

HCl: hydrochloric acid;

POCl₃ : phosphorous oxychloride;

ATP: adenosine triphosphate;

NaOH: sodium hydroxide;

TRIS-HCl: Tris(hydroxymethyl)aminomethane hydrochloride;

EDTA: ethylenediaminetetraacetic acid;

NH₄ Cl: ammonium chloride;

MgCl₂ : magnesium chloride;

CHCl₃ : chloroform;

DMSO: dimethyl sulfoxide;

NH₂ NH₂.H₂ O: hydrazine hydrate;

Me: methyl;

eq: equivalents

OER: oxygen enhancement ratio, i.e., the ratio of a radiation doserequired to reduce the survival fraction of hypoxic cells to apredetermined level (i.e., 1% of the control) compared to the radiationdose required to attain the same survival fraction in air;

SER: sensitization enhancement ratio, i.e., the ratio of the radiationdose required to reduce the survival fraction of hypoxic cells to apredetermined level (e.g., 1% of the control) compared to the radiationdose required to attain the same survival fraction with a sensitizerpresent;

C₁.6 : concentration of a sensitizer yielding an SER of 1.6;

C_(i) : intracellular concentration;

C_(e) : extracellular concentration;

C₁.6i : intracellular concentration at C₁.6 ;

IC_(50/H),1h : concentration for 50% inhibition of clonogenicity underhypoxia for 1 hour;

IC_(50/A),1h : concentration for 50% inhibition of clonogenicity underaerobic conditions for 1 hour;

ThI: therapeutic index;

IsD: isoeffective to the oxygen dose;

PC_(o/w) : partition coefficient in octanol/water;

RT: room temperature; and

TLC: thin layer chromatography.

All commercial reagents were obtained from Aldrich Co., Milwaukee, Wis.,or Eastman Kodak Co., Kingsport, Tenn. and were utilized without furtherpurification. ##STR13## wherein X=--[CH₂ ]₂ --:1a

X=--SCH₂ --:1b

X=--MeNCH₂ --:1c

X=--CH₂ --:1d,

and wherein the heteroatom in compounds 1 (b,c) is in the 2-position,whereas in original cycloketone the carbonyl is in the 4-position.##STR14##

EXAMPLE 1 Preparation of THNLA-1

The nitroimidazolylalkyl phthalimide depicted as compound 2 in the abovesynthetic scheme was prepared by first dissolving 120 mg (milligram) ofnitroimidazole in 5 ml (milliliter) dry DMSO, then slowly adding, withstirring, 42.47 mg NaH (sodium hydride, 60% dispersion in mineral oil)under a dry argon or nitrogen atmosphere to prepare the sodium salt ofnitroimidazole. After the reaction mixture became clear, 290.4 mg of3-bromopropylphthalimide (98% pure) was added in one portion to thesodium nitroimidazole solution, and the resulting mixture was stirred atroom temperature for about 24 hours. After the 3-bromopropylphthalimidewas consumed, as determined by TLC, the DMSO was removed by distillationunder reduced pressure. The resulting residue then was triturated with amethylene chloride/water (CH₂ Cl₂ /H₂ O) mixture. The resulting organiclayer was dried over sodium sulfate (Na₂ SO₄), and then filtered, andevaporated. The resulting product was identified as compound 2 by ¹ HNMR and HRMS.

Compound 2, i.e., 3-(2-nitro-1-imidazolyl)propylphthalimide, was a whitesolid obtained in a yield of about 82%. Analysis of compound 2 providedthe following data: m.p. 151°-153° C. (Mel-Temp II open capillarymelting point apparatus); ¹ H NMR (CDCl₃) δ: 2.27 (q,J=6.5 Hz, 2H); 3.78(t,J=6 Hz, 2H); 4.53 (t,J=7 Hz, 2H); 7.16 (s, 1H); 7.36 (s, 1H);7.73-7.90 (m, 4H); HRMS m/z 300.0842 calculated for C₁₄ H₁₂ N₄ O₄ ;Found: 300.0841.

Compound 3 depicted in the above synthetic sequence was prepared inaccordance with the modified hydrazinolysis method described in G. E.Adams et al., UK Pat. Appl. 2,131,020, Chemical Abstracts, 102, page6489n (1985), incorporated herein by reference. In particular, compound2 of the synthetic sequence (150 mg) and hydrazine monohydrate (25 mg,98% pure) were refluxed in 2 ml of ethanol for about 4.5 to about 5hours. The reaction mixture then was cooled and acidified with excess 1NHCl solution. Next, the acidified solution again was refluxed for 1hour, then cooled. The resulting insoluble phthalylhydrazide, aby-product, was filtered from the reaction mixture, and the ethanol wasremoved by evaporation under reduced pressure. The resulting mixture wasfiltered again to remove the remaining phthalylhydrazide, then alkalizedwith NaOH, and finally extracted ten times with CH₂ Cl₂. The organiclayer was dried over Na₂ SO₄, filtered, then evaporated under reducedpressure to provide 3-(2-nitro-1-imidazolyl)propylamine, i.e., compound3 of the synthetic sequence.

The 3-(2-nitro-1-imidazolyl)propylamine was a yellow oil which turnedorange over time, and was obtained in a yield of about 70%. Analysis ofcompound 3 provided the following data: ¹ H NMR (CDCl₃) δ: 1.35 (br,2H); 1.95 (q,J=6.96 Hz, 2H); 2.74 (t,J=6.54 Hz, 2H); 4.53 (t,J=7 Hz,2H); 7.11 (s, 1H); 7.15 (s, 1H); MS:m/z of 170 (M+).

Compound la in the synthetic scheme was prepared from anthranilic acidand cyclohexanone by heating a mixture of the compounds in POCl₃, as setforth in M. Yamato et al., J. Med. Chem., 32, pages 1295-1300 (1989),incorporated herein by reference.

Compound la (i.e., 9-chloro-1,2,3,4-tetrahydroacridine) and3-(2-nitro-1-imidazolyl)propylamine (compound 3) were admixed and heatedin PhOH (1.05 eq) and NaI (0.024 eq) at 130° C. for 15 min (minutes) ina preheated oil bath. The resulting THNLA-1 was purified by preparativeTLC (alumina, ethyl acetate, optimum yield 32%), then converted to thehydrochloride salt with HCl gas in dioxane.

The hydrochloride salt of THNLA-1 was recrystallized from anethanol:ethyl acetate mixture as a white solid, having a watersolubility of about 12mM, yield 88%. Analysis of THNLA-1 provided thefollowing data: mp 175°-180° C. (dec.); ¹ H NMR (GEN-500, 500 MHzspectrometer) (D₂ O) δ: 8.06 (d,J=8.5 Hz, 1H), 7.79 (t,J=7.8 Hz, 1H),7.65 (d,J=8.5 Hz, 1H), 7.48 (t,J =7.8 Hz, 1H), 7.32 (s, 1H), 6.99 (s,1H), 4.54 (t,J=7.0 Hz, 2H), 4.08 (t,J=6.0 Hz, 2H), 2.92 (m, 2H), 2.38(m, 4H), 1.87 (m, 4H). HRMS (VG 70-250SE mass spectrometer): Calcd forC₁₉ H₂₁ N₅ O₂ (free amine):m/z 351.1695. Found: 351.1703.

THNLA-1 was prepared as an aqueous solution and then diluted toappropriate concentrations with tissue culture medium.

EXAMPLE 2 Preparation of S-THNLA-1

Compounds 2 and 3 of the above synthetic scheme were prepared in theidentical manner described in Example 1. Compound 1b was prepared byheating anthranilic acid and tetrahydrothiopyran-4-one in POC₃ in anidentical manner as described in Example 1.

Compound 1b (i.e., 10-chloro-3,4-dihydro-1H-thiopyrano[4,3-b]quinoline)and 3-(2-nitro-1-imidazoyl)propylamine (compound 3) were reacted as setforth in Example 1 to provide S-THNLA-1. S-THNLA-1 was purified bypreparative TLC (alumina, ethyl acetate, optimum yield 27%), thenconverted to the hydrochloride salt with HCl gas in dioxane.

The hydrochloride salt of S-THNLA-1 was recrystallized from an ethanol:ethyl acetate mixture as a white solid having the water solubility ofabout 9.5 mM, yield 55%. Analysis of S-THNLA-1 provided the followingdata: ¹ H NMR (GEN-500, 500 MHz spectrometer) (D₂ O) δ: 8.06 (d,J=8.7Hz, 1H), 7.85 (t,J=8 Hz, 1H), 7.69 (d,J=8.7 Hz, 1H), 7.54 (t,J=8 Hz,1H), 7.35 (s, 1H), 6.98 (s, 1H), 4.54 (t,J=7.5 Hz, 2H), 4.04 (t,J=6.5Hz, 2H), 3.63 (s, 2H), 3.28 (t,J=6.5 Hz, 2H), 3.08 (t,J=6.5 Hz, 2H),2.40 (m, 2H). HRMS (VG 70-250SE mass spectrometer):Calcd. for C₁₈ H₁₉ N₅O₂ S (free amine) :m/z 369.125946. Found: 369.1262.

EXAMPLE 3 Preparation of MeN-THNLA-1

Compounds 2 and 3 of the above synthetic scheme were prepared in theidentical manner described in Example 1. Compound 1c was prepared byheating anthranilic acid and 1-methyl-4-piperidone in POCl₃ in anidentical manner as described in Example 1.

Compound 1c (i.e.,10-chloro-2-methyl-1,2,3,4-tetrahydro-benzo[b]-1,6-naphthyridine) and3(2-nitro-1-imidazolyl)propylamine (compound 3) were reacted as setforth in Example 1 to provide MeN-THNLA-1. MeN-THNLA-1 was purified bypreparative TLC (alumina, ethyl acetate, optimum yield 22%), thenconverted to the hydrochloride salt with HCl gas in dioxane. Thehydrochloride salt of MeN-THNLA-1 was recrystallized from anethanol:ethyl acetate mixture as a white solid having a water solubilityof about 20 mM, yield 86%. Analysis of MeN-THNLA-1 provided thefollowing data: ¹ H NMR (GEN-500, 500 MHz spectrometer) (CDCl₃) freeamine, δ: 8.00 (d,J=8 Hz, 1H), 7.91 (d,J =8 Hz, 1H), 7.66 (t,J=7 Hz,1H), 7.45 (t,J=7 Hz, 1H), 7.2 (s, 1H), 7.04 (s, 1H), 4.57 (t,J=6.8 Hz,2H), 3.67 (s, 2H), 3.55 (t,J=6 Hz, 2H), 3.28 (t,J=6 Hz, 2H), 2.88 (t,J=6Hz, 2H), 2.61 (s, 3H), 2.28 (m, 2H). FAB in m-nitrobenzyl alcohol (VG70-250SE mass spectrometer): Calcd. for C₁₉ H₂₃ N₆ O₂ (monoprotonatedform) MH+:m/z 367.1882. Found: 367.1819.

EXAMPLE 4 Preparation of NLCPQ-1

Compounds 2 and 3 of the above synthetic scheme were prepared in theidentical manner described in Example 1. Compound 1d was prepared byheating anthranilic acid and cyclopentanone in POCl₃ in an identicalmanner as described in Example 1.

Compound 1d (9-chloro-cyclopenteno[b]quinoline) and3-(2-nitro-1-imidazolyl)propylamine (compound 3) were reacted as setforth in Example 1 to provide NLCPQ-1. NLCPQ-1 was purified bypreparative TLC (alumina, ethyl acetate, yield 26%), then converted tothe hydrochloride salt with HCl gas in dioxane. The hydrochloride saltof NLCPQ-1 was recrystallized from an ethanol:ethyl acetate mixture as awhite solid having a water solubility of about 11 mM, yield 87.5%.Analysis of NLCPQ-1 provided the following data: ¹ H NMR of free amine(GEN-500, 500 MHz spectrometer) (CDCl₃), δ: 7.98 (d,J=8 Hz, 1H), 7.86(d,J=8 Hz, 1H), 7.65 (t,J=7.8 Hz, 1H), 7.48 (t,J=7.8 Hz, 1H), 7.23 (s,1H), 7.13 (s, 1H), 4.65 (t,J=7.2 Hz, 2H), 3.77 (s, br, 2H), 3.2 (t,J=6.5Hz, 2H), 3.13 (t,J=7.2 Hz, 2H), 2.32 (m, 2H), 2.21 (m, 2H). HRMS (VG70-250SE mass spectrometer) :Calcd. for C₁₈ H₁₉ N₅ O₂ (free amine) :m/z337.15387. Found: 337.1539.

S-THNLA-1, MeN-THNLA-1 and NLCPQ-1 were prepared as aqueous solutionsand then diluted to appropriate concentrations with tissue culturemedium.

As indicated above, the novel compounds of the present invention arehypoxia selective cytotoxins with improved radio- and chemosensitizingproperties. As illustrated hereafter, studies show that the presentcompounds bind less strongly to DNA through intercalation than priorsensitizers. This feature provides hypoxic sensitizers and cytotoxins ofsuperior in vitro therapeutic index compared to the fully aromatic,acridine-based NLA-series of compounds disclosed inPapadopoulou-Rosenzweig et al. U.S. Pat. No. 5,294,715.

As illustrated hereafter, the cytotoxicity, radiosensitization,chemosensitization, uptake and interactions of THNLA-1, S-THNLA-1,MeN-THNLA-1 and NLCPQ-1 with DNA and topoisomerases I or II, weredetermined using V79 Chinese hamster lung cells under both aerobic orhypoxic conditions. As also illustrated hereafter, the novel compoundsof the present invention, like THNLA-1, a 2-nitroimidazole tethered to1,2,3,4-tetrahydroacridine, are not fully aromatic compounds, and arehypoxia selective cytotoxins and radiosensitizers having a lowerDNA-binding affinity than the fully aromatic NLA-1 acridine analog. Thefully-aromatic NLA compounds are illustrated in the structural formula(XVIII) for NLA-1. The reduced DNA-binding affinity of the presentcompounds has been attributed to disruption of the planarity of thearomatic acridine ring system in the NLA series of compounds. Thedisruption of planarity also allows the present compounds to exhibit arapid extravascular diffusion in vivo and a better localization totumors. ##STR15##

Samples of V79 Chinese hamster lung cells used in the followingexperiments were prepared as follows. V79 cells, exponentially growingas monolayer cultures in RPMI 1640 medium (Mediatech) supplemented with10% fetal calf serum, were trypsinized, centrifuged for 5 min,harvested, then suspended in 25 ml Erlenmeyer flasks fitted with rubbercaps at 5×10⁵ cells/ml (5 ml). Individual flasks either were shaken (100rpm) at 37° C. under aerobic conditions or made hypoxic by gassing witha 95% N₂ (nitrogen)/5% CO₂ (carbon dioxide) humidified gas mixture for 1hour. In the various tests, a bioreductive compound of the presentinvention, such as THNLA-1, was added to a flask containing the aerobicor hypoxic V79 cells.

The acute aerobic and hypoxic cytotoxicity of THNLA-1, S-THNLA-1,MeN-THNLA-1 and NLCPQ-1 were determined by exposing samples of V79 cellsfor 1 hour at 37° C. under either hypoxic or aerobic conditions to asensitizer concentration of 0 to 0.8 mM (millimolar) (FIGS. 1B and 2),and by a one through five hour exposure to a fixed THNLA concentration(e.g., 100 μM (micromolar), FIG. 1A). To determine the fraction ofsurviving cells, samples were removed, diluted and plated to provide2×10² to 2×10⁴ cells/well on 60 mm Linbro multi-well plates (FlowLaboratories, McLean, Virginia). The plates were incubated at 37° C. for6 days, stained with crystal violet and examined for colony formation.Colonies of 50 cells or greater were counted. Each plotted point inFIGS. 1 and 2 represent the mean of three or four replicate experiments.

FIG. 1A shows that in the presence of 100 μM of THNLA-1, under aerobicconditions, a survival fraction of about 50% is observed after 3.5hours, while the corresponding hypoxic survival was less than 0.002%.FIG. 1B illustrates the THNLA-1 concentration-dependent cytotoxicityunder hypoxic/aerobic conditions after a 1 hour exposure at 37° C. FIG.1B shows that THNLA-1 is substantially more toxic to V79 cells underhypoxic conditions than aerobic conditions. For example, to achieve a10% survival fraction under hypoxic conditions, only about 0.1 mM ofTHNLA-1 is required. Under aerobic condition, about 0.475 mM of THNLA-1is required to achieve a 10% survival fraction. The compounds of thepresent invention accordingly are hypoxia selective cytotoxins.

From the experiments illustrated in FIG. 1, the IC₅₀ values(concentration for 50% inhibition of determined for THNLA-1. TheIC_(50/A),1h for THNLA-1 is clonogenicity) under hypoxia (H) or air (A)was about 360 μM and the IC_(50/H),1h is about 33 μM. Therefore, thedifferential aerobic/hypoxic toxicity (IC_(50A) /IC_(50H)) for THNLA-1is about 11. The prior art fully-aromatic NLA-1 compound appears to beabout a two times more potent hypoxic cytotoxin (IC_(50/H),1h =15 μM)than THNLA-1, however, the selectivity of NLA-1 is 5.5, or two timessmaller than THNLA-1. The in vitro therapeutic index (ThI, defined asIC_(50/A), 1h /C₁.6) for THNLA-1 and NLA-1 are about 20 and about 11,respectively.

In FIG. 2, the concentration-dependent cytotoxicity for S-THNLA-1,MeN-THNLA-1 and NLCPQ I is plotted. From this data, a differentialaerobic/hypoxic toxicity of 9, 2.2 and 8 was calculated for S-THNLA-1,MeN-THNLA I and NLCPQ-1, respectively. The corresponding ThI values forS-THNLA-1, MeN-THNLA-1 and NLCPQ-1 are about 15, 7 and 25, respectively.

Radiosensitization studies were performed on V79 cells in a mannersimilar to the acute toxicity experiments, except that the survivalfraction was determined after a predetermined radiation dose. THNLA-1was added to aerated or hypoxic V79 cells at 37° C., 1 hour beforeirradiation at room temperature (⁶⁰ Co, 1.534 Gy/min). One Gy is equalto 100 rads, wherein one rad is the quantity of ionizing radiation thatresults in the absorption of 100 ergs of energy per gram of irradiatedmaterial. Hypoxia was maintained throughout irradiation. Survival curveswere normalized for the hypoxic cytotoxicity of THNLA-1 at a zeroradiation dose (FIG. 3A). Sensitization enhancement ratios (SER) weredetermined at a 10% survival level. The C₁.6 value (i.e., aconcentration of THNLA-1 yielding an SER of 1.6) was determined byplotting SER values against THNLA-1 concentration (FIG. 3B). The plottedpoints in FIGS. 3 and 4 represent the mean of 2 or 3 replicateexperiments.

FIG. 3A shows that increasing the concentration of THNLA-1 from 0 to 100μM substantially increases the response of hypoxic V79 cells toradiation. FIG. 3B shows that the SER for THNLA is about 3 for a THNLA-1concentration of about 100 μM. The concentration dependence of the SERfor THNLA-1, S-THNLA-1 and MeN-THNLA-1 is illustrated in FIG. 4.

Significant radiosensitization of hypoxic V79 cells was observed at roomtemperature, when the THNLA-1 was administered 1 hour at 37° C. beforeirradiation. Radiosensitization was not observed under aerobicconditions. In other experiments, radiosensitization also was notobserved when THNLA-1 was administered immediately after irradiation.The degree of radiosensitization also is apparently concentrationdependent and SER values tend to approach a plateau (FIGS. 3B and 4).Accordingly, optimal doses of the hypoxia selective compound of generalstructural formula (I) can be determined. For example, exposure to 100μM THNLA-1 at 37° C. for 1 hour before irradiation at room temperaturegave an SER of about 3, which is equivalent to the OER (afternormalization for hypoxic toxicity). This concentration is notaerobically toxic (i.e., is about 28% of IC_(50/A),1h). The C₁.6 valueof THNLA is 19 μM. The C₁.6 values for S-THNLA-1, MeN-THNLA-1 andNLCPQ-1 are 40-45, 59 and 7 μM, respectively.

The isoeffective to the oxygen dose (IsD) at a constant radiation dosewas determined by exposing aerobic or hypoxic V79 cells to variableconcentrations of THNLA-1 or the prior art NLA-1 compound for 1 hour at37° C., then to a predetermined radiation dose. The survival fractionunder hypoxic conditions (plotted versus compound concentration) meetsthe aerobic survival fraction (radiation toxicity) at the IsD. The IsDvalue is a useful indicator of the overall potency of a compound as asensitizer and cytotoxin of hypoxic cells. IsD is especially useful whencomparing different compounds for hypoxic cytotoxicity andradiosensitizing efficacy.

FIGS. 5 and 6 illustrate the IsD at 7.5 Gy (FIG. 5A) and 20 Gy (FIG.5B). FIGS. 5A and 6A include a comparison to the prior art NLA-1compound. V79 cells were irradiated at 7.5 Gy or 20 Gy after a 1 hourexposure under hypoxia (symbols) or air (straight line), at various drugconcentrations. No toxicity or radiosensitization for any testedcompound was observed under aerobic conditions over the testedconcentration ranges. IsD values were calculated from the intersectionsof the survival fractions of the hypoxic curves with the survivalfraction in air. All data points in FIGS. 5 and 6 represent the mean oftwo replicate experiments.

In particular, FIGS. 5 and 6 illustrate that the IsD for THNLA-1 at 7.5Gy and 20 Gy was about 40 and about 78 μM, respectively. Thecorresponding curves obtained under hypoxic conditions for thedetermination of IsD had a different shape at the two tested radiationdoses. At the higher dose of 20 Gy, the initial shoulder, observed at7.5 Gy, disappeared. For NLA-1, the IsD at 7.5 Gy was about 11 μM. ForS-THNLA-1 and MeN-THNLA-1, the corresponding IsD at 20 Gy was about 103and about 127 μM, respectively (FIG. 6B).

The partition coefficient of the hypoxia selective cytotoxins of thepresent invention were determined by the method of T. Fujita et al., J.Am. Chem. Soc., 86, 5175-5180 (1964), incorporated herein by reference.The partition coefficient in octanol/water (PC_(o/w)) for THNLA-1 was0.14 (versus 0.07 for NLA-1). Therefore, THNLA-1 is about two times morelipophilic than NLA-1 and about two times more hydrophilic than MISO(misonidazole). However, the increased lipophilicity of THNLA-1 did notresult in an increase of the aerobic mean uptake factor(intracellular:extracellular concentration, C_(i) /C_(e)) of THNLA-1compared to NLA-1.

Intracellular (C_(i)) and extracellular (C_(e)) drug concentrations weredetermined after exposing V79 cells (2×10⁶ /ml, 5 ml) for 30 minutes at37° C. to THNLA-1 concentrations of 0 to 500 μM under aerobicconditions. Also, uptake measurements at various times under bothaerobic and hypoxic conditions were made. Afterwards, the samples werepelleted by centrifuging for 6 minutes at 0° C. A small volume of thesupernatant (200 μl ) was combined with 9 equal volumes (1.8 ml) ofacetonitrile and the remaining supernatant was discarded. The sampleswere centrifuged again to remove residual supernatant and the pelletswere lysed with 90 μl water and deproteinized with 0.9 ml acetonitrile.After centrifugation and filtration, all samples were stored at -70° C.until a UV spectroscopic analysis was made at two different wavelengths: 330 nm (nanometers) (absorption of the nitro-group) and 244 nm(λ_(max)). Samples including only V79 cells were treated in an identicalmanner for correction of the measurements, while a calibration curve wasobtained by measuring the absorption of known concentrations of THNLA-1in the same solvent system. Mean intracellular concentration of drug wascalculated using a value of 810 fl as an intracellular water content oflog-phase cells.

FIG. 7A illustrates a comparison of mean intracellular (C_(i)) andextracellular (C_(e)) concentrations (in μM) of THNLA-1 over an inputTHNLA-1 concentration of 0 to 500 μM. FIG. 7B illustrates uptake factors(i.e., C_(i) /C_(e)) under aerobic conditions for 30 minutes at 37° C.over the same THNLA-1 input concentrations. FIG. 7C illustrates theuptake of THNLA-1 (20 μM) by aerobic and hypoxic V79 cells after a 15,30 or 60 minute incubation time at 37° C. Concentrations were determinedby UV spectrophotometry at 330 nm. The plotted points represent resultsfrom two experiments, in triplicate.

Changes in the intracellular accumulation of THNLA-1, as well as thecorresponding extracellular concentration, by increasing the inputconcentration of THNLA-1 is illustrated in FIG. 7A. FIG. 7A shows thatthe intracellular concentration (C_(i)) of THNLA-1 is substantiallygreater than the corresponding extracellular concentration (C_(e)) ofTHNLA-1. FIG. 7B shows that the uptake factor (C_(i) /C_(e)) reaches amaximum at an input concentration of about 100 μM THNLA-1 and thenstarts to decrease. FIG. 7C illustrates that under hypoxic or aerobicconditions, the C_(i) of THNLA-1 is substantially greater than theC_(e).

In particular, the C_(i) of THNLA-1 remained basically unchanged over15, 30 and 60 minutes incubation periods under aerobic conditions, whilea decrease of C_(i) over time was observed under hypoxic conditions(FIG. 7C). This decrease is attributed to metabolism under hypoxicconditions. The intracellular concentration for an SER of 1.6 (C₁.6i)for THNLA-1 and NLA-1 are 0.443 and 0.885 mM, respectively.

The PC_(o/w) for S-THNLA-1, MeN-THNLA-1 and NLCPQ-1 were measured as0.41, 0.40, and 0.30, respectively, showing the increased lipophilicityof S-THNLA-1, MeN-THNLA-1 and NLCPQ-1 over NLA-1. A comparison of theaerobic uptake by V79 cells for THNLA-1, S-THNLA-1, MeN-THNLA-1 andNLCPQ-1 is shown in FIG. 8.

Uptake measurements in the presence of the lysosomotropic agent ammoniumchloride (NH₄ Cl) also were performed in a manner as previouslydescribed. Ammonium chloride was added to the V79 cell samples atdifferent concentration levels 15 minutes prior to THNLA-1 addition. TheV79 cells then were incubated with 60 μM of THNLA-1 (or NLA-1) for 45minutes under aerobic conditions at 37° C.

FIG. 9 illustrates the effect of ammonium chloride on C_(i) and C_(e)concentration of THNLA-1 (FIG. 9A) or NLA-1 (FIG. 9B) at 60 μM inputconcentration in aerobic V79 cells. FIG. 9C compares the effect ofammonium chloride addition on the C_(i) accumulation of THNLA-1 andNLA-1 at 60 μM input concentration in aerobic V79 cells. Concentrationswere determined by UV spectrophotometry at 330 nm. The plotted pointsrepresent the mean of two experiments.

When V79 cells were incubated for 15 minutes with increasingconcentrations of NH₄ Cl prior to THNLA-1 addition and under aerobicconditions, the intracellular concentration (C_(i)) of THNLA-1 wasdecreased significantly (i.e., a factor of about 3, at 50 mM NH₄ Cl,FIG. 9A). The C_(i) for the prior art compound NLA-1 also decreased byabout a factor of 3 (FIG. 9B). A comparison of the aerobic uptake of theTHNLA-1 and NLA-1 in the presence of NH₄ Cl is shown in FIG. 9C. Asimilar decrease in aerobic uptake was observed for S-THNLA-1 in thepresence of NH₄ Cl (FIG. 10A). A comparison of the effect of NH₄ Cl onthe aerobic uptake of THNLA-1, S-THNLA-1 and NLA-1 is depicted in FIG.10B.

FIG. 11A illustrates the uptake reduction rates for THNLA-1 and NLA-1(in μM per mM of NH₄ Cl) over an increasing NH₄ Cl concentration range.FIG. 11B illustrates the correlation between C₁.6 values andcorresponding C_(i) /C_(e) values for NLA-1, THNLA-1, S-THNLA-1 andMeN-THNLA-1.

An agarose gel electrophoresis technique was used to determine whetherTHNLA-3 intercalates DNA. Compounds that strongly intercalate DNA retardDNA mobility during agarose gel electrophoresis due to unwinding andextension of the dsDNA. Agents that do not intercalate DNA, or that bindto DNA via other mechanisms, do not decrease DNA mobility in this assay.

In particular, a Topo I drug screening kit (TopoGEN, Inc., Columbus,Ohio) was used to assay the topoisomerase I THNLA-1 interaction. Thetests were performed in a 20 μl (final volume) solution containing:water, assay buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 100 mM NaCl),0.75 μg of supercoiled plasmid DNA, 6 units of calf thymus Topo I (in 50mM Tris.HCl, pH 7.55, 0.7 M (molar) Nacl, 0.5 mM EDTA, 0.5 mMdithiothreitol, 10% glycerol) and various concentrations of THNLA-1 or100 μM camptothecin for comparison. Camptothecin is a Topoisomerase Ipoison used as an anticancer drug. Camptothecin stabilizes the TopoI-DNA clearable complex, and therefore prevents the resealing of DNA byTopo-I.

Individual reaction n mixtures were heated for 30 minutes at 37° C. on aheating block, and then 2 μl of 10% SDS-proteinase K solution was addedto each reaction mixture. The resulting reaction mixtures then wereheated for an additional 30 minutes at 37° C. The reaction mixtures in aloading buffer were extracted with CHCl₃ prior to submarineelectrophoresis on a 1% agarose gel. The DNA bands were observedvisually by staining with ethidium bromide (0.5 μg/ml).

In the THNLA-1 -topoisomerase I interaction test, supercoiled (sc) DNAwas incubated either alone, in the presence of Topo I (2 units), or inthe presence of Topo I (6 units) and inhibitor, i.e., camptothecin (100μM) or THNLA-1 (100, 200, 400, 600, 800 or 1000 μM). Other incubatedsamples were scDNA and THNLA-1 (1000 μM) in the absence of Topo I, bothunextracted and after extraction with CHCl₃. The results of thetopoisomerase I test clearly showed that THNLA-1 inhibits Topo I-inducedrelaxation of supercoiled DNA, but at significantly higherconcentrations than NLA-type compounds.

A Topo II assay kit (TopoGEN, Inc., Columbus, Ohio) also was used toassay the topoisomerase II -THNLA-1 interaction. The tests wereperformed on a 20 μl (final volume) solution containing: water, cleavagebuffer (30 mM Tris.HCl, pH 7.6, 3 mM ATP, 15 mM 2-mercaptoethanol, 8 mMMgCl₂, 60 mM NaCl), 0.3 μg (microgram) of kinetoplast DNA [KDNA], 4units of human Topo II and various concentrations of THNLA-1. Individualreaction mixtures were heated for 30 minutes at 37° C. on a heatingblock, and then 2 μl of a 10% SDS-proteinase K solution was added toeach reaction mixture. The resulting reaction mixtures then were heatedfor an additional 30 minutes at 37° C. After addition of a 0.1 volume ofloading buffer, the reaction mixtures were extracted with CHCl₃ prior tosubmarine electrophoresis on a 1% agarose gel containing ethidiumbromide (0.5 μg/ml).

In the THNLA-1-Topoisomerase II interaction test, kinetoplast DNA (KDNA)was incubated either alone, in the presence of Topo II (4 units) or inthe presence of Topo II and inhibitor, i.e., THNLA-1 (200, 400, 600, 800or 1000 μM, respectively). Other incubated samples were KDNA and THNLA-1(1000 μM) in the absence of Topo II, a linear KDNA marker, and adecatenated KDNA marker. The results of the Topoisomerase II assayclearly showed that THNLA-1 does not inhibit the Topo II-induceddecatenation of KDNA even at very high concentrations, contrary to theNLA-compounds.

Contrary to the strong DNA-intercalating NLA-compounds, THNLA-1,S-THNLA-1 and MeN-THNLA-1 do not affect the mobility of supercoiled DNAon the electrophoretic gel up to millimolar concentrations. Accordingly,the hypoxia selective cytotoxins of the present invention have beenshown to bind less efficiently to DNA. Even though THNLA-1 initiatesinhibition of supercoiled DNA relaxation, induced by Topo I, at 100 μMconcentration, complete inhibition was not observed at THNLA-1concentrations even up to 1 mM. The observed inhibition is attributed toeither unwinding of DNA through intercalation or direct interaction withthe Topo I. THNLA-1 also did not inhibit decatenation of kinetoplast DNA[KDNA] induced by topoisomerase II up to a 1 mM concentration ofTHNLA-1.

S-THNLA-1 and MeN-THNLA-1 also did not inhibit the action ofTopoisomerase II on kinetoplast DNA up to a 1 mM concentration. Theaction of Topoisomerase I on closed circular DNA was completelyinhibited when about 800 μM of S-THNLA-1 was used. In addition, somenicked DNA also was observed. However, S-THNLA-1 alone also caused theformation of some nicked DNA at a concentration of 800 μM. MeN-THNLA-1,in concentrations of up to 1000 μM, only partially inhibited Topo I, andno formation of nicked DNA was observed.

The physicochemical and biological properties of THNLA-1 are summarizedand compared to NLA-1 in Table I. The physicochemical arid biologicalproperties of S-THNLA-1, MeN-THNLA-1 and NLCPQ-1 are summarized in TableII.

FIGS. 2, 4, 6, 8 and 10 illustrate that S-THNLA-1, Me-THNLA-1 andNLCPQ-1 possess properties similar to THNLA-1 with respect toconcentration dependent cytotoxicity (FIG. 2), SER (FIG. 4), IsD (FIG.6), V79 cell uptake (FIG. 8) and effect of ammonium chloride (FIG. 10).

                  TABLE I                                                         ______________________________________                                        A Comparison of Physicochemical Properties,                                   Biological Activities and Drug Uptake in                                      V79 Cells (37° C.) between THNLA-1 and NLA-1                           Property              THNLA-1   NLA-1                                         ______________________________________                                        Partition Coefficient in                                                                            0.14      0.07                                          octanol/water:PC.sub.o/w                                                      Aerobic Cytotoxicity:IC.sub.50/A,1h                                                                 360.sup.a)                                                                              84                                            Hypoxic cytotoxicity:IC.sub.50/H,1h                                                                 33        15                                            Hypoxic selectivity:IC.sub.50/A,1h /IC.sub.50/H,1h                                                  11        5.5                                           Hypoxic radiosensitizing potency:C.sub.1.6                                                          19        8                                             Intracellular concentration at C.sub.1.6 :C.sub.1.6i                                                443       885                                           Therapeutic index (ThI):IC.sub.50/A,1h /C.sub.1.6                                                   20-30     11                                            Isoeffective to the oxygen dose at 7.5                                                              40        11                                            Gy:IsD.sub.(7.5)                                                              Topo I inhibition (50%)-dose:                                                                       about 600 about 12                                      ______________________________________                                         .sup.a) All concentrations are in μM.                                 

                  TABLE II                                                        ______________________________________                                        Physicochemical Properties, Biological Activities                             and Drug Uptake in V79 Cells (37° C.)                                  for S-THNLA-1, MeN-THNLA-1 and NLCPQ-1                                        Property   S-THNLA-1  MeN-THNLA-1  NLCPQ-1                                    ______________________________________                                        Partition Co-                                                                            0.40       0.41         0.30                                       efficient in                                                                  octanol/water:                                                                PC.sub.o/w                                                                    Aerobic Cyto-                                                                            580.sup.a) 426          170                                        toxicity:IC.sub.50/A,1h                                                       Hypoxic cyto-                                                                            68         196          22                                         toxicity:IC.sub.50/H,1h                                                       Hypoxic selec-                                                                           about 9    about 2.2    about 8                                    tivity:IC.sub.50/A,1h /                                                       IC.sub.50/H,1h                                                                Hypoxic radio-                                                                           40         59           7                                          sensitizing                                                                   potency:C.sub.1.6                                                             Intracellular                                                                            932        586          209                                        concentration at                                                              C.sub.1.6 :C.sub.1.6i                                                         Therapeutic index                                                                        about 15   about 7      about 25                                   (ThI):IC.sub.50/A,1h /                                                        C.sub.1.6                                                                     Isoeffective to the                                                                      60         nd           25.5                                       oxygen dose at 7.5                                                            Gy:IsD.sub.(7.5)                                                              Topo I inhibition                                                                        about 800  nd           nd.sup.b)                                  (50%)-dose:                                                                   ______________________________________                                         .sup.a) All concentrations are in μM.                                      .sup.b) Not determined.                                                  

As previously stated, hypoxic tissues are resistant to radiation therapyand chemotherapeutic drugs. The resistance to chemotherapy is attributedto the distance of the target from viable blood vessels, a slower rateof proliferation, and to the hypoxic environment itself. In addition Losensitizing hypoxic cells to radiation treatment, the bioreductive drugsof the present invention also have shown a therapeutic gain whencombined with chemotherapeutic alkylating agents. It has beenhypothesized, but not relied upon herein, that the therapeutic gain is aresult of potentiating alkylating agent-induced DNA crosslinks bymetabolites of nitroimidazole.

To demonstrate the usefulness of the compounds of structural formula(I), chemosensitization studies were performed with THNLA-1 usingmelphalan (i.e., L-PAM) or cis-DDP as the chemotherapeutic agents.L-PAM, also known as phenylalanine mustard and available from SigmaChem. Co., St. Louis, Mo., first was dissolved in an ethanolic solutionof HCl (0.5N). The ethanolic HCl solution was buffered with propyleneglycol in a 1:9 ratio of ethanolic HCl solution to propylene glycol, andfinally diluted 100 fold with suspension medium. cis-DDP, aplatinum-based chemotherapeutic agent available from Sigma Chem. Co.,St. Louis, Mo., was dissolved in water to a predetermined concentration.

To examine the "preincubation effect" of the compounds of generalformula (I), V79 cells were exposed to a fixed concentration of THNLA-1for 2 hours under hypoxic conditions, followed by exposure to varyingconcentrations of L-PAM or cis-DDP for 1 hour under aerobic conditionsat 37° C., and then assayed for colony formation. In evaluating theeffect of "preincubation time" on chemosensitization, V79 cells wereexposed to fixed concentrations of THNLA-1 under conditions of hypoxiafor 0 to 4 hours at 37° C., followed by exposure to L-PAM (2 μg/ml) orcis-DDP (30 μM) under aerobic conditions for 1 hour at 37° C., and thenassayed for colony formation. THNLA-1 dose-dependent potentiation alsowas examined by exposing V79 cells to various THNLA-1 concentrations for2 hours at 37° C. under hypoxic conditions, and then to a fixed dose ofeach chemotherapeutic agent for 1 hour at 37° C. under aerobicconditions. Experiments using a simultaneous addition of the sensitizerand chemotherapeutic agent for 1 hour at 37° C. under aerobic conditionswere also performed.

In all experiments, controls for the hypoxic cytotoxicity of thesensitizer and the toxicity of the chemotherapeutic alone were included.Survival curves were normalized for the hypoxic cytotoxicity ofsensitizer in order to determine the dose modification factor (DMF),i.e., the ratio of chemotherapeutic agent concentrations required toreduce cell survival to a predetermined level (e.g., 0.5) alone or incombination with a sensitizer. Synergism between the chemotherapeuticagent and the chemosensitizer was determined using the fractionalproduct concept which is applied in instances of independent action ofdrugs, or by isobologramic analysis. The results of thechemosensitization studies are illustrated in FIGS. 12-15.

With further respect to chemosensitization, isobologramic analysis andanalysis according to the fractional product concept clearlydemonstrated a synergistic interaction between THNLA-1 and eachchemotherapeutic agent (e.g., L-PAM and cis-DDP) under hypoxicpretreatment conditioning of the V79 cells. The synergistic effect isrelated to the hypoxiapretreatment time with THNLA-1 (FIG. 12), onTHNLA-1 concentration (FIG. 13), and the concentration of thechemotherapeutic drug (FIG. 14). The DMF values of L-PAM and cis-DDP at0.5 survival fraction are 3.2 and 4.2, respectively, when 10 or 15 μM ofTHNLA-1 was used, respectively. Isobolograms for a survival fraction of0.316 are shown in FIG. 15.

With further respect to FIGS. 12 and 13, the dashed lines represent theexpected additive effect of combining THNLA-1 with either L-PAM orcis-DDP. In FIG. 12, the V79 cell samples were exposed to THNLA-1 (5 =M)under hypoxic conditions for a time period of one to four hours prior toa one hour aerobic exposure to either L-PAM or cis-DDP. In FIG. 13, theV79 cell samples were exposed to a THNLA-1 concentration of 0 to 40 mMfor 2 hours under hypoxic conditions prior to one hour aerobic exposureto L-PAM Dr cis-DDP. The survival fraction was observed, and illustratedthe synergistic effect of combining THNLA-1 with each chemotherapeuticcompound tested.

FIG. 14 illustrates a dramatic decrease in survival fraction when afixed amount THNLA-1 is combined with L-PAM or cis-DDP over a wideconcentration range. The V79 cells were exposed to 10 or 15 μM ofTHNLA-1 for two hours under hypoxic conditions prior to aerobic exposureto L-PAM or cis-DDP for one hour, respectively.

The isobolograms of FIG. 15 clearly show the synergistic effect ofcombining THNLA-1 with L-PAM or cis-DDP. If the effect of combiningTHNLA-1 with L-PAM or cis-DDP was purely additive, tile plotted pointswould fall in the zone within the curves labeled Mode I and Mode II,i.e., the envelope of additivity. If the combination of THNLA-1 andL-PAM or cis-DDP is antagonistic, the plotted points would fall in thezone to the right of the envelope of additivity. In an isobologram, likeFIG. 15, a combination is synergistic when the plotted points fall tothe left of the envelope of additivity. Accordingly, the combination ofTHNLA-1 and L-PAM or cis-DDP exhibits an unexpected synergistic effect.The plotted points in FIG. 15 represent a treatment using THNLA-1 andL-PAM or cis-DDP producing a survival fraction of 0,316.

THNLA-1 therefore is a powerful potentiator of chemotherapeutic agenttoxicity against V79 cells. Isobologramic and fractional product conceptanalysis showed that synergistic interaction occurs between THNLA-1 andthe chemotherapeutic agents L-PAM and cis-DDP under hypoxic pretreatmentconditions. The magnitude of the synergistic effect is related tohypoxia pretreatment time with THNLA-1, to THNLA-1 concentration, and tothe concentration of the chemotherapeutic drug. A longer hypoxicpreexposure time and a lower sensitizer dose are preferred over ashorter hypoxic preexposure time and a higher sensitizer dose. Otherexperiments showed that THNLA-1 also effectively chemosensitizes OVCARcells to L-PAM and cis-DDP. OVCAR cells are resistant to L-PAM andcis-DDP.

Tests also were performed using S-THNLA-1 or NLCPQ-1 with L-PAM orcis-DDP. Those tests showed that S-THNLA-1 and NLCPQ-1, like THNLA-1,are strong chemosensitizers of V-79 cells to L-PAM and cis-DDP.

In accordance with an important feature of the present invention, theusefulness of the hypoxia selective cytotoxins having structural formula(I) as radiation therapy and chemotherapy sensitizers has beendemonstrated. Although THNLA-1 is about a two times less potentcytotoxin of hypoxic cells than the prior art NLA-1 compound (i.e.,IC_(50/H),1h :33 versus 15 μM), THNLA-1 is about four times lesscytotoxic under aerobic conditions (IC_(50/A),1h :360 versus 82 μM) and,therefore, is about two times more selective towards hypoxic V79 cellsthan NLA-1 (11 versus 5.5). This improved hypoxia selectivity has beenattributed to a decrease in aerobic toxicity mediated by mechanismsindependent of bioreduction. The decreased toxicity of THNLA-1 underhypoxic conditions compared to NLA-1 can be a result of differences inbioreduction rates in combination with differences in uptake factors.

THNLA-1 also has a superior therapeutic index (ThI) compared to NLA-1.As a radiosensitizer of hypoxic cells, THNLA-1 is about two times morepotent than NLA-1 on the basis of the C₁.6i values (C₁.6i :0.443 versus0.885 mM, respectively), even though comparison of only C₁.6 values leadto opposite conclusions (C₁.6 :19 versus 8 μM, respectively). Similarbehavior has been observed in the case of 5-nitroquine (5-NQ) and1-nitracrine (1-NC), two prior art compounds that are structurallyrelated to THNLA-1 and NLA-1, respectively. Also, based on the C₁.6ivalues, THNLA-1 is a more potent radiosensitizer of hypoxic cells thanmisonidazole and the weak base pimonidazole (0.70 and 0.69 mM,respectively). A maximum SER value of 3.1 was achieved with THNLA-1(e.g., 100 μM, 28% of IC_(50/A),1h) because of the low toxicity ofTHNLA-1.

The in vitro therapeutic indices (ThI) for S-THNLA-1 and MeN-THNLA-1 arenot significantly different from the therapeutic index of the prior artNLA-1 (about 15 and about 7, respectively, versus 11). S-THNLA-1, whichis more hypoxia selective, also has a better therapeutic index thanNLA-1. S-THNLA-1 is a better hypoxia selective cytotoxin than the fullyaromatic NLA-1 (having a differential toxicity of about 9 versus 5.5,respectively). MeN-THNLA-1 has a differential toxicity of 2.2,indicating that for MeN-THNLA-1 the binding affinity to DNA is notcorrelated to differential toxicity.

NLCPQ-1, S-THNLA-1 and MeN-THNLA-1 also are very efficientradiosensitizers with maximum SER values (equal to OER) at non-toxicconcentrations. However, on a concentration basis S-THNLA-1 andMeN-THNLA-1 appear less potent than NLA-1 and THNLA-1, which isattributed to a different uptake by V79 cells. However, based on C₁.6ivalues, MeN-THNLA-1 is a more potent radiosensitizer than NLA-1, misoand pimonidazole.

NLCPQ-1, on the other hand, has a potency as a radiosensitizer that issimilar to NLA-1 (C₁.6 of 7 for NLCPQ-1, C₁.6 of 8 for NLA-1). The ThIof NLCPQ-1 is 25, which is superior to the THI of 11 exhibited by NLA-1.Based again on the C₁.6i values on Table II, NLCPQ-1 is 4.2 times morepotent than NLA-1, and is the most potent of all the tested compounds asa radio- or chemosensitizer and as a hypoxia selective cytotoxin.

No radiosensitization was observed under hypoxic conditions when THNLA-1was given immediately after radiation. Therefore, a hypoxia selectivecytotoxin of the present invention should be present at the target siteat time of irradiation. In addition, it was observed that noradioprotection occurred under aerobic conditions. The prior art NLA-1compound has afforded radioprotection under aerobic conditions. Theaerobic radioprotection provided by NLA-1 helps explain the failure ofNLA-1 as radiosensitizer in solid tumors where not all regions of thetumor are hypoxic. Aerobic radioprotection also has been observed withphenanthridium compounds. In accordance with another important featureof the present invention, the ability to radiosensitize under hypoxicconditions, and not to radioprotect under aerobic conditions,illustrates an important potential clinical advantage of the hypoxiaselective cytotoxins having general structural formula

With respect to chemosensitization, the DMF values obtained for acombination of cis-DDP or L-PAM with THNLA-1 were slightly greater thanthe DMF value obtained using a combination including NLA-1 (using about30% of the IC_(50/H),1h). However, based on the intracellularconcentration level (C_(i)) of the two compounds, THNLA-1 is asignificantly more efficacious chemosensitizer than NLA-1. The fact thatno potentiation occurred when cells were exposed to THNLA-1 and L-PAM orcis-DDP under aerobic conditions is further indicative that no systemictoxicity due to THNLA-1 should be expected in vivo.

The relatively high THNLA-1 concentrations required for Topo-Iinhibition (600 μM versus 12 μM for NLA-1) helps explain theunexpectedly low aerobic toxicity of THNLA-1. Furthermore, inhibition ofthe catalytic activity of Topo-I without induction of Topo I-dependentsingle-stranded DNA cleavage is not lethal to the cell. Such cleavage isnot observed with THNLA-1, while it has been observed with NLA-1.

In conclusion, the DNA-affinic compounds of the present invention are animprovement over the prior acridine-based NLA compounds as hypoxiaselective cytotoxins, radiosensitizers and chemosensitizes. For example,THNLA-1 has a two-to-three times greater in vitro therapeutic index thanNLA-1. In vivo tests using THNLA-1, S-THNLA-1, MeN-THNLA-1 and NLCPQ-1are designed to illustrate the usefulness of the new and improvedbioreductive drugs in potential clinical use.

FIGS. 16 and 17 illustrate in vivo radiosensitization tests usingTHNLA-1 (FIG. 16) or etanidazole (FIG. 17), a prior sensitizer, eitheralone or in combination with a single radiation dose (20 Gy). FIGS. 16Aand 16B illustrate replicate sets of experiments wherein THNLA-1 wasadministered to balb mice bearing two EMT6 tumors. The THNLA-1 wasadministered at a concentration of 0.103 mmol/g, intraperitoneally,either 60, 45 ,or 30 minutes prior to irradiation at 20 Gy. EMT6 tumor:sexhibit up to 20% hypoxia.

FIGS. 16A and 16B also show the toxicity of THNLA-1 alone (short-dashedline). FIG. 16B shows the toxicity of radiation (20 Gy) alone (solidline). FIGS. 16A and 16B also illustrate the expected additive effect ofradiation and THNLA-1 (long-dashed line and dot-dash line in FIGS. 16Aand 16B, respectively), and illustrate the synergistic effect ofcombining radiation with a pretreatment of THNLA-1.

A toxicity study performed on balb mice showed that THNLA-1 is nontoxicat a concentration of at least 0.129 mmol/g. Therefore the dose of 0.103mmol/g used in the experiment illustrated in FIG. 16 can be increasedfor optimum sensitivity. THNLA-1 also exhibits a potent radiosensitizingeffect at 0.103 mmol/g in vivo. Finally, the degree of radiosensitivityis related to the amount of time lapsing between THNLA-1 administrationand subsequent radiation. FIGS. 16A and 16B particularly show thatradiosensitization is maximized when the THNLA-1 is administered atleast one hour before irradiation.

FIGS. 17A-C illustrate experiments similar to those illustrated in FIGS.16A-B, except the radiosensitizer was the prior art compound,etanidazole (i.e., SR-2508), administered at 2 mmol/g. A comparisonbetween FIG. 16 and FIG. 17 shows that THNLA-1, at 0.103 mmol/g,enhanced the effects of 20 Gy radiation in EMT6 tumors to a similardegree as 2 mmol/g of etanidazole, i.e., THNLA-1 performs essentiallyequally as a radiosensitizer to etanidazole at about one-twentieth ofthe dose.

Obviously, many modifications and variations of the invention ashereinbefore set forth can be made without departing from the spirit andscope thereof and therefore only such limitations should be imposed asare indicated by the appended claims.

We claim:
 1. A hypoxia selective cytotoxin having the structuralformula: ##STR16## wherein ##STR17## is selected from the groupconsisting of ##STR18## R₁ and R₂, independently, are selected from thegroup consisting of methyl, halo, hydrogen, trifluoromethyl, methoxy,cyano, and methylsulfo; R₃ and R₄ taken together are a five orsix-membered nonaromatic ring system; n is an integer 1 through 5; X iscarbon or nitrogen; and Z is a physiologically acceptable anion.
 2. Thecytotoxin of claim 1 wherein R₃ and R₄ are taken together to form a fiveor six-membered nonaromatic carbocyclic ring.
 3. The cytotoxin of claim1 wherein R₃ and R₄ are taken together to form a five or six-memberednonaromatic ring incorporating an oxygen atom, a sulfur atom or anitrogen atom.
 4. The cytotoxin of claim 1 wherein R₃ and R₄, when takentogether to form a five or six-membered non-aromatic ring, is selectedfrom the group consisting of --(CH₂)₃ --, --CHMe(CH₂)₂ --, --CH₂ CHMeCH₂--, --(CH₂ )₂ CHMe--, --(CH₂ )₄ --, --CHMe(CH₂)₃ --, --CH₂ CHMe(CH₂)₂--, --(CH₂)₂ CHMeCH₂ --, --(CH₂)₃ CHMe--, --SCH₂ CH₂ --, --CH₂ SCH₂ --,--CH₂ SCH₂ CH₂ --, --CH₂ OCH₂ --, --CH₂ OCH₂ CH₂ --, --CH₂ NR₅ CH₂ CH₂--, --(CH₂)₃ NR₅ -- and --(CH₂)₂ NR₅ --, wherein Me is methyl and R₅ ismethyl or ethyl.
 5. The cytotoxin of claim 1 wherein n is an integer twothrough four.
 6. The cytotoxin of claim 1 wherein ##STR19##
 7. Thecytotoxin of claim 1 wherein HZ is selected from the group consisting ofhydrochloric acid, phosphoric acid, nitric acid, perchloric acid,tetrafluoroboric acid, sulfuric acid, and mixtures thereof.
 8. A methodof chemosensitization comprising administering an effective amount ofthe hypoxia selective cytotoxin of claim 1 to a tumor or tumor cells,then administering a chemotherapeutic agent.
 9. The method of claim 8wherein the chemotherapeutic agent is selected from the group consistingof L-PAM, cis-DDP, cyclophosphamide, a nitrosourea, and doxorubicin. 10.A hypoxia selective cytotoxin having the structural formula: ##STR20##wherein ##STR21## is selected from the group consisting of ##STR22## R₁and R₂, independently, are selected from the group consisting of methyl,halo, hydrogen, trifluoromethyl, methoxy, cyano, and methylsulfo; R₃ andR₄ taken together are a five or six-membered nonaromatic ring system;and n is an integer 1 through 5; X is carbon or nitrogen.
 11. A hypoxiaselective cytotoxin having the structural formula: ##STR23## wherein D,E, F, and G, independently, are carbon or nitrogen, with the provisothat three or more of D, E, F, and G are carbon; R₁ is methyl; R₂ ismethyl; R₃ and R₄ taken together are a five- or six-membered nonaromaticring system; n is an integer 1 through 5; X is carbon or nitrogen, and Zis a physiologically acceptable anion.
 12. A hypoxia selective cytotoxinhaving the structural formula: ##STR24## wherein D, E, F, and G,independently, are carbon or nitrogen, with the proviso that three ormore of D, E, F, and G are carbon; R₁ is fluoro; R₂ is fluoro; R₃ and R₄taken together are a five- or six-membered nonaromatic ring system; n isan integer 1 through 5; X is carbon or nitrogen, and Z is aphysiologically acceptable anion.
 13. A hypoxia selective cytotoxinhaving the structural formula: ##STR25## wherein D, E, F, and G,independently, are carbon or nitrogen, with the proviso that three ormore of D, E, F, and G are carbon; R₁ is methoxy; R₂ is methoxy; R₃ andR₄ taken together are a five- or six-membered nonaromatic ring system; nis an integer 1 through 5; X is carbon or nitrogen, and Z is aphysiologically acceptable anion.
 14. A hypoxia selective cytotoxinhaving the structural formula: ##STR26## wherein D, E, F, and G,independently, are carbon or nitrogen, with the proviso that three ormore of D, E, F, and G are carbon; R₁ is chloro; R₂ is chloro; R₃ and R₄taken together are a five- or six-membered nonaromatic ring system; n isan integer 1 through 5; X is carbon or nitrogen, and Z is aphysiologically acceptable anion.
 15. A hypoxia selective cytotoxinselected from the group consisting of9-[3-(2-nitro-1-imidazolyl)propylamino]-cyclopenten[b]quinolinehydrochloride, having the structure: ##STR27##
 16. A method ofchemosensitization comprising administering an effective amount of ahypoxia selective cytotoxin to a tumor or tumor cells, said hypoxiaselective cytotoxin selected from the group consisting of9-[3-(2-nitro-1-imidazolyl)propylamino]cyclopenten[b]quinolinehydrochloride, having the structure: ##STR28## then administering achemotherapeutic agent.